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Separation and purification of the mRNAs for vesciular stomatitis virus NS and M proteins
VIROLOGY
98, 251-254 (1979)
Separation
KEVIN
and Purification
R. LYNCH,*
of the mRNAs for Vesicular Stomatitis NS and M Proteins
DIANE AND PAUL
Roche
Institute
*University
PENNICA,’ HERBERT S. COHEN*
of Molecular Biology, Nutley, of Rhode Island, Kingston, Accepted
July
New Rhode
Virus
L. ENNIS,
Jersey, 07110, and Island, 02881
20, 1979
All five vesicular stomatitis virus mRNAs were separated and purified by acid-urea agarose gel electrophoresis. The identities of the NS and M mRNAs were established by their % vitro translation.
Vesicular stomatitis virus (VSV) infected cells contain, in addition to 42 S genome RNA, five discrete viral messenger species designated L, G, N, NS, and M (1-3). The L, G, and N messenger RNAs have been resolved both by formamide-polyacrylamide gel electrophoresis (1, 3) and sucrose density gradient centrifugation (1). However, the VSV NS and M messages, both of which are present in the 12 S gradient fraction, have only been resolved as duplexes with the viral genome (&,5>. The individual NS and M messages, however, have not been purified. In the present study, we were able to separate and purify the 12 S VSV messenger species into the M and NS mRNAs without prior duplex formation. Acid-urea agarose gels provide excellent resolution of RNA molecules under denaturing conditions (6), since separation is based both on molecular sieving and on the different charge to mass ratios assumed by different RNA molecules at an acid pH. Optimum resolution of VSV mRNAs was achieved on a 30 em, 1.25% agarose slab gel at pH 3.8, using 3H-labeled RNA isolated from VSV-infected cells at 3 h after infection (Fig, 1A). Only the bottom onethird of the autoradiogram is shown since this demonstrates the relevant information. It is noteworthy that all of the VSV [“H]mRNAs, including the two 12 S species, are resolved using this gel system. The ’ To whom reprint request should be addressed. 251
genome and L RNAs were found at approximately 8 and 13 cm, respectively, from the top of the gel (data not shown). In order to detect maximum separation, we used 3H-labeled RNA instead of 32P-labeled VSV RNA since resolution of the RNA species is not apparent due to the autoradiographic spread characteristic of 32P. To insure that the conditions employed were sufficiently denaturing, we used two criteria to test the homogeneity of the major bands shown in Fig. 1A. First, the chemical purity of the bands was determined by extracting each of the VSV RNAs from the gel and electrophoresing the separated bands on a second urea-agarose gel. As illustrated in Fig. lB, the six major VSV RNA species contained no apparent contamination. Second, these VSV RNAs were translated in a wheat-germ cell-free protein synthesizing system (Fig. 2). As can be seen, the slower migrating component of the 12 S RNA was the message for the VSV M protein (Fig. 2, lane 9), and the faster migrating 12 S component directed the synthesis of the NS protein (Fig. 2, lane 8). Thus, the order of migration of the VSV M and NS mRNAs was the reverse of that reported when VSV RNAs were separated as duplexes on polyacrylamide gels (4, 5). Figure 2 further demonstrates that the 14.5 S RNA is the mRNA for the N protein (lane 7). The synthesis of the G protein is directed by the 17 S RNA although small amounts of 0042-6822/79/130251-04$02.00/O Copyright All rights
0 1979 by Academic Press, Inc. of reproduction in any form reserved.
252
SHORT COMMUNICATIONS
A
17s 14.5 s ’12s
FIG. 1. (A) Separation of VSV RNAs by acid-urea agarose gel electrophoresis. Total 3H-labeled RNA isolated from VSV-infected CHO cells at 3 hr after infection was resuspended in 6 M urea, 0.025&f Na-citrate pH 3.8, and heated at 37°C for 20 min before layering on the gel. Gels were prepared by adding 1.25 g Seakem agarose (Marine Colloids) to a solution containing ‘75ml of deionized 8 M urea, 10 ml 0.25 M Na-citrate pH 3.8, and 15 ml sterile H,O (final pH approximately 4.5). The agarose was dissolved by boiling for 15 min. After cooling, slab gels (0.15 x 15 x 30 cm) were cast in a Hoffer slab gel apparatus (Bio-Rad Laboratories) and the agarose was allowed to solidify over-
SHORT COMMUNICATIONS 123456789 FIG. 2. Translation of VSV mRNAs extracted from acid-urea agarose gels. Each of the RNAs analyzed in the gel shown in Fig. 1B was used to program a wheat-germ cell-free protein synthesizing system under conditions of ribosome excess. The translation products were separated on a sodium dodecyl sulfate10% polyacrylamide gel (II) and fluorographed as described by Laskey and Mills (9). Translation parameters have been described elsewhere (8). Individual lanes contain translation products of reactions programmed with the following RNA species: Lane 1, ‘7 pg total VSV RNA isolated from CHO cells at 3 hr after infection; lane 2, no RNA; lane 3, same as lane 1; lane 4, 42 S RNA; lane 5, 30 S RNA; lane 6, 17 S RNA, lane 7, 14.5 S RNA; lane 8, 12 S RNA (fast component); lane 9, 12 S RNA (slow component). Lane 1 represents a longer exposure (24 hr) than the other lanes (5 hr) in order to reveal the translation of the VSV L protein. RNA extracted from dried agarose gels was also functionally active in vitro (data not shown).
N and NS proteins were also observed (lane 6). The same result had been shown previously using RNA isolated from formamide-polyacrylamide gels (7). It is possible that the G mRNA is contaminated with small amounts of NS and N messages. Another interesting explanation for this observation
M
is that the 17 S mRNA may contain, in addition to G mRNA, unprocessed, polycistronic RNA species, (also approximately 17 S), coding for both the N and NS proteins and corresponding to the 3’ end of the VSV genome RNA. The 42 S (genome RNA) and 30 S
night at 4°C. Gels were run in the cold room at 4°C at a constant current of 25 mA with recirculation of a buffer consisting of 0.025 M Na-citrate, pH 3.8. Electrophoresis was stopped when the brom phenol blue tracker dye had migrated out of the gel (approximately 19 hr). The gels were fluorographed as described by Laskey and Mills (9). This figure represents the bottom one-third of the gel since it contains all the relevant information and since we did not have the facilities to dry the entire gel intact. Details concerning culturing of virions and CHO cells, labeling of VSV RNAs, and RNA extraction have been discussed elsewhere (8). The letters on the left denote the VSV gene product directed by each mRNA (see Fig. 2). (B) Electrophoresis of isolated VSV RNAs extracted from acid-urea agarose gels. 3ZP-labeled VSV total RNA isolated from CHO cells at 3 hr after infection was separated on a 1.5% agarose slab gel (0.15 x 15 x 13 cm) at pH 3.5. Gels were prepared and run as described in the legend to (A), except that the RNA was heated at 37°C for 20 min in 90% DMSO before layering on the gel (10). The change in agarose concentration and pH from the gel described in (A) did not have any effect on the relative positions of the individual RNA species. An autoradiogram prepared from the wet gel was used as a template when cutting out the bands. Since resolution of the 12 S species was obscured by autoradiographic spread, only the leading and trailing 25% of the large 12 S band was cut. The bands containing 42,30, 17, 14.5, and both 12 S viral RNAs were cut from the gel, ground in a Ten Broeck homogenizer and the agarose was removed by centrifugation. This procedure was repeated and 50 Kg/ml Escherichia coli tRNA was added to the combined supernatants before precipitating with 2 vol of ethanol. The final pellets were dissolved in sterile water and dialyzed against water, then each of the isolated RNA species was electrophoresed on a second agarose gel which is shown in this figure. Preparation of s2P-labeled VSV RNAs was performed as described by Rose and Knipe (8). The far left lane represents total “2P-labeled VSV RNA. The numbers on the right of the gel are approximate S values determined from VSV RNAs isolated from sucrose density gradients after velocity sedimentation (data not shown).
254
SHORT
COMMUNICATIONS
(presumably the L protein mRNA) did not direct the synthesis of any detectable proteins (lanes 4 and 5). Although we were able to translate only four of the five mRNAs after their extraction from the agarose gels, we have previously shown (8), that all five viral proteins were synthesized when total RNA from VSVinfected cells was used to direct the in vitro system (Fig. 2, lanes 1 and 3). It is apparent from our data that the molecular weight of the NS protein as estimated by SDS-polyacrylamide gels is much larger than that expected from a messenger that appears equal in size to the M protein mRNA. It should be emphasized that the relative rates of migration of the M and NS mRNAs do not necessarily reflect relative sizes, because at acid pH migration is a function of differences in both size and charge to mass ratios (6). However, since the M and NS mRNAs can now be purified, their molecular weights can be accurately determined by direct chemical methods. In summary, these data demonstrate that acid-urea agarose gel electrophoresis is a simple, rapid method for the separation and
subsequent isolation of all five VSV mRNAs under denaturing conditions. REFERENCES 1. BOTH, G. W., MOYER, S. A., and BANERJEE, A. K., Proc. Nat. Acad. Sci. USA 72, 274278 (1975). 2. MORRISON, T., STAMPFER, M., BALTIMORE, D., and LODISH, H. F., J. Viral. 13, 62-72 (1974). 3. ROSE, J. K., and KNIPE, D., J. Viral. 15, 9941003 (197.5). 4. FREEMAN, G. J., ROSE, J. K., CLINTON, G. M., and HUANG, A. S., J. Viral. 21, 1094-1104 (1977). 5. RHODES, D. P., ABRAHAM, G., COLONNO, R. J., JELINEK, W., and BANERJEE, A. K., J. Viral. 21, 1105-1112 (19’77). 6. LEHARCH, H., DIAMOND, D., WOZNEY, J. M., and BOEDTKER, H., Biochemistry 16, 4743-4751 (1977). 7. KNIPE, D., ROSE, J. K., and LODISH, H. F., J. Viral. 15, 1004-1011 (1975). 8. PENNICA, D., LYNCH, K. R., COHEN, P. S., and ENNIS, H. L., Virology 94, 484-487 (1979). 9. LASKEY, R. A., and MILLS, A. D., Eur. J. Biochem. 56, 335-341 (1975). 10. CLINTON, G. M., LISLE, S. P., HAGEN, F. S., and HUANG, A. S., Cell 15, 1455-1462 (1978). 11. ENNIS, H. L., PENNICA, D., and HILL, J. M., Develop. Biol. 65, 251-259 (1978).
98, 251-254 (1979)
Separation
KEVIN
and Purification
R. LYNCH,*
of the mRNAs for Vesicular Stomatitis NS and M Proteins
DIANE AND PAUL
Roche
Institute
*University
PENNICA,’ HERBERT S. COHEN*
of Molecular Biology, Nutley, of Rhode Island, Kingston, Accepted
July
New Rhode
Virus
L. ENNIS,
Jersey, 07110, and Island, 02881
20, 1979
All five vesicular stomatitis virus mRNAs were separated and purified by acid-urea agarose gel electrophoresis. The identities of the NS and M mRNAs were established by their % vitro translation.
Vesicular stomatitis virus (VSV) infected cells contain, in addition to 42 S genome RNA, five discrete viral messenger species designated L, G, N, NS, and M (1-3). The L, G, and N messenger RNAs have been resolved both by formamide-polyacrylamide gel electrophoresis (1, 3) and sucrose density gradient centrifugation (1). However, the VSV NS and M messages, both of which are present in the 12 S gradient fraction, have only been resolved as duplexes with the viral genome (&,5>. The individual NS and M messages, however, have not been purified. In the present study, we were able to separate and purify the 12 S VSV messenger species into the M and NS mRNAs without prior duplex formation. Acid-urea agarose gels provide excellent resolution of RNA molecules under denaturing conditions (6), since separation is based both on molecular sieving and on the different charge to mass ratios assumed by different RNA molecules at an acid pH. Optimum resolution of VSV mRNAs was achieved on a 30 em, 1.25% agarose slab gel at pH 3.8, using 3H-labeled RNA isolated from VSV-infected cells at 3 h after infection (Fig, 1A). Only the bottom onethird of the autoradiogram is shown since this demonstrates the relevant information. It is noteworthy that all of the VSV [“H]mRNAs, including the two 12 S species, are resolved using this gel system. The ’ To whom reprint request should be addressed. 251
genome and L RNAs were found at approximately 8 and 13 cm, respectively, from the top of the gel (data not shown). In order to detect maximum separation, we used 3H-labeled RNA instead of 32P-labeled VSV RNA since resolution of the RNA species is not apparent due to the autoradiographic spread characteristic of 32P. To insure that the conditions employed were sufficiently denaturing, we used two criteria to test the homogeneity of the major bands shown in Fig. 1A. First, the chemical purity of the bands was determined by extracting each of the VSV RNAs from the gel and electrophoresing the separated bands on a second urea-agarose gel. As illustrated in Fig. lB, the six major VSV RNA species contained no apparent contamination. Second, these VSV RNAs were translated in a wheat-germ cell-free protein synthesizing system (Fig. 2). As can be seen, the slower migrating component of the 12 S RNA was the message for the VSV M protein (Fig. 2, lane 9), and the faster migrating 12 S component directed the synthesis of the NS protein (Fig. 2, lane 8). Thus, the order of migration of the VSV M and NS mRNAs was the reverse of that reported when VSV RNAs were separated as duplexes on polyacrylamide gels (4, 5). Figure 2 further demonstrates that the 14.5 S RNA is the mRNA for the N protein (lane 7). The synthesis of the G protein is directed by the 17 S RNA although small amounts of 0042-6822/79/130251-04$02.00/O Copyright All rights
0 1979 by Academic Press, Inc. of reproduction in any form reserved.
252
SHORT COMMUNICATIONS
A
17s 14.5 s ’12s
FIG. 1. (A) Separation of VSV RNAs by acid-urea agarose gel electrophoresis. Total 3H-labeled RNA isolated from VSV-infected CHO cells at 3 hr after infection was resuspended in 6 M urea, 0.025&f Na-citrate pH 3.8, and heated at 37°C for 20 min before layering on the gel. Gels were prepared by adding 1.25 g Seakem agarose (Marine Colloids) to a solution containing ‘75ml of deionized 8 M urea, 10 ml 0.25 M Na-citrate pH 3.8, and 15 ml sterile H,O (final pH approximately 4.5). The agarose was dissolved by boiling for 15 min. After cooling, slab gels (0.15 x 15 x 30 cm) were cast in a Hoffer slab gel apparatus (Bio-Rad Laboratories) and the agarose was allowed to solidify over-
SHORT COMMUNICATIONS 123456789 FIG. 2. Translation of VSV mRNAs extracted from acid-urea agarose gels. Each of the RNAs analyzed in the gel shown in Fig. 1B was used to program a wheat-germ cell-free protein synthesizing system under conditions of ribosome excess. The translation products were separated on a sodium dodecyl sulfate10% polyacrylamide gel (II) and fluorographed as described by Laskey and Mills (9). Translation parameters have been described elsewhere (8). Individual lanes contain translation products of reactions programmed with the following RNA species: Lane 1, ‘7 pg total VSV RNA isolated from CHO cells at 3 hr after infection; lane 2, no RNA; lane 3, same as lane 1; lane 4, 42 S RNA; lane 5, 30 S RNA; lane 6, 17 S RNA, lane 7, 14.5 S RNA; lane 8, 12 S RNA (fast component); lane 9, 12 S RNA (slow component). Lane 1 represents a longer exposure (24 hr) than the other lanes (5 hr) in order to reveal the translation of the VSV L protein. RNA extracted from dried agarose gels was also functionally active in vitro (data not shown).
N and NS proteins were also observed (lane 6). The same result had been shown previously using RNA isolated from formamide-polyacrylamide gels (7). It is possible that the G mRNA is contaminated with small amounts of NS and N messages. Another interesting explanation for this observation
M
is that the 17 S mRNA may contain, in addition to G mRNA, unprocessed, polycistronic RNA species, (also approximately 17 S), coding for both the N and NS proteins and corresponding to the 3’ end of the VSV genome RNA. The 42 S (genome RNA) and 30 S
night at 4°C. Gels were run in the cold room at 4°C at a constant current of 25 mA with recirculation of a buffer consisting of 0.025 M Na-citrate, pH 3.8. Electrophoresis was stopped when the brom phenol blue tracker dye had migrated out of the gel (approximately 19 hr). The gels were fluorographed as described by Laskey and Mills (9). This figure represents the bottom one-third of the gel since it contains all the relevant information and since we did not have the facilities to dry the entire gel intact. Details concerning culturing of virions and CHO cells, labeling of VSV RNAs, and RNA extraction have been discussed elsewhere (8). The letters on the left denote the VSV gene product directed by each mRNA (see Fig. 2). (B) Electrophoresis of isolated VSV RNAs extracted from acid-urea agarose gels. 3ZP-labeled VSV total RNA isolated from CHO cells at 3 hr after infection was separated on a 1.5% agarose slab gel (0.15 x 15 x 13 cm) at pH 3.5. Gels were prepared and run as described in the legend to (A), except that the RNA was heated at 37°C for 20 min in 90% DMSO before layering on the gel (10). The change in agarose concentration and pH from the gel described in (A) did not have any effect on the relative positions of the individual RNA species. An autoradiogram prepared from the wet gel was used as a template when cutting out the bands. Since resolution of the 12 S species was obscured by autoradiographic spread, only the leading and trailing 25% of the large 12 S band was cut. The bands containing 42,30, 17, 14.5, and both 12 S viral RNAs were cut from the gel, ground in a Ten Broeck homogenizer and the agarose was removed by centrifugation. This procedure was repeated and 50 Kg/ml Escherichia coli tRNA was added to the combined supernatants before precipitating with 2 vol of ethanol. The final pellets were dissolved in sterile water and dialyzed against water, then each of the isolated RNA species was electrophoresed on a second agarose gel which is shown in this figure. Preparation of s2P-labeled VSV RNAs was performed as described by Rose and Knipe (8). The far left lane represents total “2P-labeled VSV RNA. The numbers on the right of the gel are approximate S values determined from VSV RNAs isolated from sucrose density gradients after velocity sedimentation (data not shown).
254
SHORT
COMMUNICATIONS
(presumably the L protein mRNA) did not direct the synthesis of any detectable proteins (lanes 4 and 5). Although we were able to translate only four of the five mRNAs after their extraction from the agarose gels, we have previously shown (8), that all five viral proteins were synthesized when total RNA from VSVinfected cells was used to direct the in vitro system (Fig. 2, lanes 1 and 3). It is apparent from our data that the molecular weight of the NS protein as estimated by SDS-polyacrylamide gels is much larger than that expected from a messenger that appears equal in size to the M protein mRNA. It should be emphasized that the relative rates of migration of the M and NS mRNAs do not necessarily reflect relative sizes, because at acid pH migration is a function of differences in both size and charge to mass ratios (6). However, since the M and NS mRNAs can now be purified, their molecular weights can be accurately determined by direct chemical methods. In summary, these data demonstrate that acid-urea agarose gel electrophoresis is a simple, rapid method for the separation and
subsequent isolation of all five VSV mRNAs under denaturing conditions. REFERENCES 1. BOTH, G. W., MOYER, S. A., and BANERJEE, A. K., Proc. Nat. Acad. Sci. USA 72, 274278 (1975). 2. MORRISON, T., STAMPFER, M., BALTIMORE, D., and LODISH, H. F., J. Viral. 13, 62-72 (1974). 3. ROSE, J. K., and KNIPE, D., J. Viral. 15, 9941003 (197.5). 4. FREEMAN, G. J., ROSE, J. K., CLINTON, G. M., and HUANG, A. S., J. Viral. 21, 1094-1104 (1977). 5. RHODES, D. P., ABRAHAM, G., COLONNO, R. J., JELINEK, W., and BANERJEE, A. K., J. Viral. 21, 1105-1112 (19’77). 6. LEHARCH, H., DIAMOND, D., WOZNEY, J. M., and BOEDTKER, H., Biochemistry 16, 4743-4751 (1977). 7. KNIPE, D., ROSE, J. K., and LODISH, H. F., J. Viral. 15, 1004-1011 (1975). 8. PENNICA, D., LYNCH, K. R., COHEN, P. S., and ENNIS, H. L., Virology 94, 484-487 (1979). 9. LASKEY, R. A., and MILLS, A. D., Eur. J. Biochem. 56, 335-341 (1975). 10. CLINTON, G. M., LISLE, S. P., HAGEN, F. S., and HUANG, A. S., Cell 15, 1455-1462 (1978). 11. ENNIS, H. L., PENNICA, D., and HILL, J. M., Develop. Biol. 65, 251-259 (1978).